Structural insights into the disulfide isomerase and chaperone activity of TrbB of the F plasmid type IV secretion system

Bacteria have evolved elaborate mechanisms to thrive in stressful environments. F-like plasmids in gram-negative bacteria encode for a multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbBWT, resulting in a truncation construct TrbB37-161, does not affect its catalytic in vitro activity compared to the wild-type protein (p = 0.76). Residues W37–K161, which include the active thioredoxin motif, are sufficient for DI activity. The N-terminus of TrbBWT is disordered as indicated by a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and 1H–15N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy of the untagged protein. This disordered region likely contributes to the protein's dynamicity; removal of this region results in a more stable protein based on 1H–15N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbBWT in the presence of TraW, a T4SSF assembly protein predicted to interact with TrbBWT, does not support the inference of a stable complex forming in vitro. This work advances our understanding of TrbB's structure and function, explores the role of structural disorder in protein dynamics in the context of a T4SSF accessory protein, and highlights the importance of redox-assisted protein folding in the T4SSF.


Introduction
Bacteria utilize a multitude of mechanisms to survive in stressful and competitive environments, including mechanisms that can harm other cells.Secretion systems (SSs) are large multi-protein complexes expressed by bacteria that aid in their survival due to their numerous functionalities (Costa et al., 2015;Filloux, 2022;Green and Mecsas, 2016;Nicholson and Champion, 2022).SSs can breach a target cell's lipid membrane(s), allowing them to perform acts of host pathogenesis by mediating the secretion of toxins and virulence genes, enhancing bacterial survivability, attaching to host cells, eliminating target cells and thus outcompeting the host cell for resources in the immediate environmental niche.There are a total of eleven types of multi-protein SSs found in the bacterial kingdom, some of which are categorized into subtypes and each classified based on the pathway of secretion and the type of secreted macromolecule (Costa et al., 2015;Trivedi et al., 2022).Some SSs, such as Type III and Type VI, mediate inter-microbial competition through the injection of effector proteins from the cytoplasm directly across host membranes (Allsopp et al., 2020;Cianfanelli et al., 2016;Trivedi et al., 2022).Other SSs, such as Type IV SS, mediate the export of both proteins and DNA from the cytosol of T4SS-containing bacteria to either a prokaryotic or a eukaryotic host cell, and it is central in the import of extracellular DNA or a toxic protein (Beck et al., 2014;Bragagnolo et al., 2020;Grace et al., 2019;Trivedi et al., 2022).In gram-negative bacteria bearing F-like plasmids, the Type IV SS (T4SS F ) is functional for bacterial proliferation and adaptation through the process of conjugation, a process that is also clinically relevant as it arbitrates the spread of antibiotic resistance genes among bacteria.
In gram-negative T4SSs encoded by F-like plasmids (F, R100, pSLT, pED208, and pYJ016), the trbB and traF genes are two of six genes that have no orthologues in P-or I-like subfamilies, and each protein contains a thioredoxin motif, a structural domain consisting of a minimum of three α-helices flanking a four-stranded antiparallel β-sheet (Bragagnolo et al., 2020;Collet and Bardwell, 2002;Elton et al., 2005;Frost et al., 1994;Kinch et al., 2003;Lawley et al., 2003;Missiakas and Raina, 1997).The TraF thioredoxin-like domain does not contain the characteristic CXXC active site, and TraF's function in T4SS F pilus extension, independent of redox activity, remains unclear.TrbB contains an active thioredoxin domain with a CXXC active site (Audette et al., 2004;Collet and Bardwell, 2002;Elton et al., 2005;Frost et al., 1994).
TrbB has been shown to function as a DI in vitro and in vivo (Elton et al., 2005;Frost et al., 1994;Hemmis et al., 2011;Lawley et al., 2003), suggesting that TrbB may ensure proper protein folding of T4SS F proteins.Several T4SS F proteins are remarkable for their high cysteine content and these proteins are putative client proteins for TrbB; TraN,TraU,and TraH each have 22,11, and 6 cysteines, respectively (Bragagnolo et al., 2020;Frost et al., 1994;Hemmis et al., 2011).Additionally, TraW, another T4SS F protein functional in pilus assembly (Bragagnolo et al., 2020;Maneewannakul and Maneewannakul, 1992), was previously identified as a binding partner for TrbB (Harris and Silverman, 2004).Conjugative T4SSs express TrbB alone, or TraF/TrbB; non-redox active TraF alone is not observed (Bragagnolo et al., 2020;Elton et al., 2005).For instance, the F and pED208 plasmids contain both TraF and TrbB while the R27 has only a redox active TraF, also suggesting different roles for TraF and TrbB within the T4SS F conjugative apparatus.Here, we report solution-based biophysical and computational structural analyses, advancing our understanding of TrbB's structure, enzymatic activity, and its interaction with other T4SS F proteins.

Cloning, protein expression and purification
Cloning of trbB 37-161 into the vector pGEX-4T-2 was done by PCR amplification from the T4SS F -containing pOX38 plasmid (Anthony et al., 1994).The forward primer used was 5′-GTACTGAATT-Caatggttccgtctcagtaatggcagg-3'; the reverse primer used was 5′-GTCTTGTCGACttatttcgcaccttttttcctccgtacatctgc-3' (Integrated DNA Technologies).Amplicon and vector were double digested with EcoRI and SalI, ligated in vitro, and constructs were transformed into Escherichia coli by heat shock.Cloning was verified by colony PCR, mini-prepping plasmids from colonies followed by double-digestion with restriction enzymes, and by Sanger sequencing at The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON) using standard pGEX primers supplied by the institution.Other expression plasmids, encoding for GST-TrbB WT (aa 21-161), His 6 TraW, and His 6 ΔTraW (aa 51-210) were from lab stocks.

Circular Dichroism (CD) spectroscopy
CD spectra were acquired between 200 and 260 nm using a Jasco J810 CD spectrometer at 22 • C and a protein concentration of 5 μM in 10 mM KH 2 PO 4 pH 7.5, 100 mM kF, 5% (v/v) glycerol to minimize noise (Greenfield, 2007).Following acquisition, CD spectra were input into BeStSel (Micsonai et al., 2022) for secondary structure content prediction.Parameters include units of measured ellipticity (mdeg) at 5 μM, the number of residues (TrbB 37-161 : 125, TrbB WT : 161), and the pathlength (0.1 cm).Thermal denaturation measurements were performed by sampling from a single wavelength (222 nm for α-helices) as a function of temperature (30-90 • C) at a rate of 1 • C/min.All CD experiments were performed in triplicate, and the mean of the internal replicates are reported.Thermal denaturation spectra data points are mean of three internal replicates with the standard deviation represented as error bars.

1 H-15 N Heteronuclear Single Quantum Correlation (HSQC) Nuclear Magnetic Resonance (NMR) spectroscopy
TrbB 37-161 and TrbB WT were expressed from E. coli BL21(DE3) grown in M9 minimal media (6 g of Na 2 HPO 4 , 3 g of KH 2 PO 4 , 1 g of 15 NH 4 Cl, 0.5 g of NaCl, and 10 g of glucose in 1 L of water supplemented with 1 mM CaCl 2 , 1 mM MgSO 4 , 50 μg/mL kanamycin, and a trace mineral mix).Large-scale protein expression and purification was done as described in Section 2.1.Purified protein samples were concentrated to 0.2 mM and supplemented with 10% (v/v) D 2 O in 10 mM HEPES pH 6.0, 50 mM NaCl.HSQC spectra were acquired on a Bruker DRX 600 NMR spectrometer operating at a 1 H frequency of 599.80 MHz at 21 • C.

SEC-MALS small angle X-ray scattering
Size exclusion chromatography linked to multi-angle light scattering and small angle X-Ray scattering (SEC-MALS-SAXS) data for GST-TrbB WT was collected at the BioCAT 18ID beamline (Advanced Photon Source, Argonne National Laboratory, USA).The protein sample was prepared without GST cleavage as per Section 2.1 and was buffer exchanged into a 10X dilution of a buffer stock (20 mM HEPES pH 7.0, 100 mM NaCl, 5% [v/v] glycerol, 0.05% NP40) using a 30 kDa MWCO concentrator.The 5.5 mg/mL GST-TrbB WT sample (as quantified via the Edelhoch method; Edelhoch, 1967;Gill and von Hippel, 1989;Pace et al., 1995) and the matching buffer were sent to the BioCAT facility where the buffer was diluted 10X and used as the running buffer for the SEC-MALS-SAXS experiment.GST-TrbB WT was injected at a volume of 350 μL onto a Superdex 200 10/300 Increase SEC column (Cytiva) using an Agilent Infinity II HPLC at 0.6 mL/min, and the sample underwent sequential multi-angle laser light scattering analysis using a Wyatt DAWN Heleos II MALS system; protein concentration was determined in-line with a refractive index detector.SAXS data was acquired using an Eiger2 XE 9M detector at a sample-detector distance of 3.7 m and at λ = 0.1033 nm.Data processing was performed using BioXTAS RAW v.2.1.1 and ATSAS packages (Hopkins et al., 2017;Manalastas-Cantos et al., 2021).Ab initio reconstruction was performed using DAMMIF (30 independent runs) with DAMAVER averaging and refinement using the ColabFold-AlphaFold2 (CF-AF2) GST-TrbB WT model, and clustering using DAMCLUST.Subsequently, the Ensemble Optimization Method (EOM) was employed to gain further insight into the structural disorder of the protein system (Bernadó et al., 2007;Tria et al., 2015).The experimental SAXS data was utilized to generate a pool of predicted models in various conformations from which a selected representative ensemble was used to determine the protein's degree of conformational polydispersity.The program was accessed through ATSAS Online (Gajoe version 2.1) (Petoukhov et al., 2007) where EOM settings were configured to 20 maximum number of curves per ensemble, 5 minimum number of curves per ensemble, with constant subtraction and curve repetition in the ensemble allowed, 100 cycles of the genetic algorithm run, and 10,000 theoretical curves.The assigned protein parameters that provided the best model were when the protein was designated as monomer with residues 1-218 as compact, 219-271 as disordered, and 272-396 as compact.The program CORAL (complexes with random loops) was also employed via ATSAS online to determine whether the SAXS data better fits a monomeric or dimeric construct by validating models of input rigid bodies with constrained positionalities connected by a generated disordered linker (Petoukhov et al., 2012).Models of the GST monomer and the TrbB 37-161 monomer from CF-AF2 were used as the input for the domains in modeling the GST-TrbB monomer with CORAL; a linker of 46 residues (10 disordered residues belonged to the GST tag) was specified between the C-terminal of the GST model and the N-terminal of the TrbB 37-161 model.Angular units were set to 1/Å based on the input SAXS data and symmetry was set to P1.In modeling the GST-TrbB dimer, multiple CORAL experiments were performed where two copies of each model with the linker were specified either with no contacts, or with contact conditions and domain groupings for a GST dimeric interface, a TrbB 37-161 dimeric interface, and both GST and TrbB 37-161 interfaces.Bead models were visualized using PyMOL v2.5.2 (Schrödinger Inc.), where the bead radius was set to 1.5 for TrbB's N-term and 5.4 for the rest (based on the size set by DAMMIF).CF-AF2 predicted models were also aligned with the SAXS-EOM bead models using PyMOL v.2.5.2.SAXS data for GST-TrbB WT were deposited to the SASBDB (https://www.sasbdb.org);accession code: SASDTL5.

Fluorometric disulfide isomerase in vitro assay
PDI activity was assessed using a fluorometric protein disulfide isomerase quenched-fluorophore kit from Abcam (Boston, USA; cat ab273337).TrbB WT and TrbB 37-161 were buffer exchanged into the kit's PDI assay buffer supplied by the manufacturer prior to analyses.Signal detection was performed using a Synergy H4 Microplate reader (Agilent BioTek) with excitation and emission wavelengths of 490 nm and 580 nm, respectively, at 25 • C. Data were collected in triplicate (n = 3) from two independently expressed and purified protein samples with final concentrations 50 μM in each well.Statistical t-tests were performed using GraphPad Prism v.9.5.1 (Dotmatics).

Analytical UPLC Size-Exclusion Chromatography
TrbB WT , TrbB 37-161 , His 6 TraW, and His 6 ΔTraW at 19 μM in 20 mM HEPES pH 7.5, 150 mM NaCl, with or without 2 mM DTT, were analyzed using a Zenix SEC-150 column (Sepax Tech.Inc.) at a rate of 1.0 mL/min on an H-class Ultra Performance Liquid Chromatography (UPLC) system (Waters Acquity) with A 280 sample detection, and sample peaks were compared to a Gel Filtration Standard of proteins with known MW and elution profiles (Bio-Rad;No. 1511901) to qualitatively gauge sample size and oligomeric state.The column was washed with 1 column volume of buffer (15 mL of 20 mM HEPES pH 7.5, 150 mM NaCl) prior to every protein analysis.The TrbB WT /His 6 TraW, TrbB WT /His 6 ΔTraW, TrbB 37-161 /His 6 TraW mixtures were equilibrated on ice for at least 30 min prior to SEC analysis to allow for any complex formation.TrbB WT / His 6 ΔTraW was analyzed to determine whether the deletion of the flexible N-terminus of TraW would affect the observation of a complex.Samples were also reduced in 2 mM Dithiothreitol (DTT), including TrbB, and analyzed with SEC.
Wild-type TrbB (TrbB WT ) was predicted by ColabFold-AlphaFold2 (CF-AF2) to have a disordered N-terminal domain (Fig. 1A, B; Supplementary Fig. S1).This disordered region comprises about a quarter of the wild-type protein (Fig. 1A), contributes to its flexibility, and likely plays a role in its interaction with multiple substrate proteins as a disulfide isomerase (DI) (Arhar et al., 2021).The TrbB 37-161 construct was designed to remove this disordered region (Fig. 1A-C); whether this region is involved in substrate binding is unclear, though it is not required for disulfide isomerase activity (Section 3.3), and the truncated protein is a more stable construct.CD spectroscopy of TrbB WT and TrbB 37-161 indicate modest changes at the secondary structure level between the wild-type and truncated forms (Fig. 1D).The percentage of α-helical content incrementally decreases from 6.2% in TrbB WT to 6.1% TrbB 37-161 (Fig. 1E).β-Sheet composition modestly decreases from 35.4% in TrbB WT to 33.7% TrbB 37-161 , respectively (Fig. 1E).The thermal denaturation of TrbB 37-161 indicates that, while it is comprised of only 6.1% α-helix, it is a stable construct with a T M of 78 • C (Fig. 2A).The predicted model (Fig. 1C) organizes the α-helices in an amphipathic fashion, with 43.8% of helix-forming residues being polar (25.0%) or charged (18.7%) (Supplementary Fig. S2), fostering a stable packing of the helices within the thioredoxin-like motif.
Conversely, the thermal denaturation profile of TrbB WT is strikingly different from TrbB 37-161 and inconsistent across replicates (Fig. 2B), making it difficult to ascertain its T M precisely.Thus, while the two constructs have very similar secondary structures, only TrbB 37-161 is thermally stable.We infer that the wild-type protein fails to have a consistent thermal denaturation profile because of the generally disordered and dynamic N-terminal region, making it less stable and more susceptible to aggregation.
TrbB WT and TrbB 37-161 were characterized with 1 H-15 N Heteronuclear Single Quantum Correlation (HSQC) Nuclear Magnetic Resonance (NMR) spectroscopy (Fig. 3).A significant distinction between TrbB WT and TrbB 37-161 was observed; the signals arising from the wildtype protein are evidently less resolved whereas the signals from the truncation mutant are smaller, concentric, and more resolved.Peak assignment and structure solution is more feasible for TrbB 37-161 due to its resolved peaks (Fig. 3).Two inferences can be drawn from the signals between 8 and 8.5 ppm in the 1 H dimension (x-axis).First, TrbB WT (magenta) is partially disordered, as the clustering of signals in this region corresponds to partial disorder in a protein (Kwan et al., 2011;Marion, 2013).Removal of the N-terminus in TrbB 37-161 (black) results in better peak resolution, indicative of a protein with a more well-defined fold.The signal differences between the two samples indicate that the removal of the N-terminal region resulted in a thermally and structurally stable TrbB 37-161 construct.While the two constructs have very similar secondary structures (according to CD, Fig. 1D and E), the NMR of TrbB WT is distinct from TrbB 37-161 (Fig. 3) because the presence or absence of the N-terminus creates distinct intramolecular environments.Together, the similar secondary structures indicated by CD and the distinct NMR of TrbB WT versus TrbB 37-161 suggest that the N-terminus does not affect the protein's secondary structures, but its flexibility and dynamicity allow it to occupy different locales of the protein in a transient manner.The transient spatial proximity of the N-terminus to a particular locale of the protein at a given moment alters the magnetic environment of that part of the protein, resulting in the observed crosspeak overlaps (Fig. 3, magenta) (Forsberg et al., 2023;Schiavina et al., 2024).Overall, given that thermally and structurally stable proteins are generally more amenable to crystallization (Derewenda, 2004;McPherson and Gavira, 2014), TrbB 37-161 is a better construct for ongoing crystallization efforts.
We investigated whether there are differences between empirical and computational methods with respect to estimating secondary structures for TrbB WT .Comparing computational data from CF-AF2 and empirical data from CD spectroscopy, differences in percentage composition between the two methods are evident (Fig. 1E, F).The CF-AF2 predicted model of TrbB WT indicates a secondary structure composition of 34.8% α-helix and 22.40% β-sheet (Fig. 1F).In contrast, CD spectroscopy empirically determined the secondary structure content at 6.2% α-helix and 35.4% β-sheet (Fig. 1E).In the TrbB 37-161 truncation mutant, CF-AF2 predicts protein composition of 38.4% α-helix and 28.0% β-sheet (Fig. 1F); CD spectroscopy indicates 6.1% α-helix and 33.7% β-sheet (Fig. 1E).Comparing the CF-AF2 predicted models of the wild-type protein (Fig. 1B) and the truncated construct (Fig. 1C) shows the removal of the predominantly disordered region (Supplementary Fig. S1, dotted circle).Therefore, the increase in composition of α-helix and β-sheet in TrbB 37-161 can be attributed to the decrease in overall size of the protein, given that the percentage composition shown in Fig. 1F was calculated by normalizing the number of residues predicted to form α-helices or β-sheets by the total sequence of the protein.Differences between CF-AF2 and empirical CD data highlight the importance of utilizing a diverse set of tools in a post-AlphaFold era for efficient hypotheses-testing and problem solving (Terwilliger et al., 2023).
Intrinsically disordered and/or highly dynamic regions are increasingly becoming appreciated as functional moieties in proteins, highlighted by discoveries of their function in interactomes (Norris et al., 2023;Schlessinger et al., 2007).These disorder-based interactions are gaining more attention and are being found to transiently bind a diverse set of partner proteins at high specificity (Sharma et al., 2015;Uversky, 2011;Wright and Dyson, 2015).Furthermore, these proteins tend to self-associate and form stable aggregates (Uversky, 2010).Interestingly, the formation of stable protein aggregates, such as the accumulation of aggregated Tau in neuronal cells, is clinically relevant as it is a hallmark of Alzheimer's Disease (Irvine et al., 2008;Mochizuki et al., 2018;Trejo-Lopez et al., 2022).Lastly, comparative studies have shown that intrinsically disordered proteins (IDPs) or regions (IDRs) generally have low hydrophobicity and exhibit large net charge at physiological pH (Babu, 2016;Uversky et al., 2000).Based on CF-AF2, we hypothesized that the N-terminal region of TrbB WT , which is about a quarter of TrbB WT (Fig. 1A), behaves as a disordered region.The high isoelectric point (pI 10.7) of the N-terminal region (S1-R36), suggests that it has a large net positive charge at physiological pH, supporting the proposal of its intrinsic disorder (Babu, 2016;Uversky et al., 2000).This hypothesis can explain why TrbB WT forms stable aggregates at concentrations above 5 mg/mL, making it difficult to study using methods that require a considerable protein concentration such as SEC-MALS-SAXS or X-Ray crystallography.

TrbB WT is highly dynamic partly due to its disordered N-terminus
TrbB WT was further analyzed using SEC-MALS-SAXS.TrbB WT is highly prone to aggregation at concentrations above 5 mg/mL, so the GST-tagged construct was utilized for this experiment to increase its solubility, making it amenable to SEC-MALS-SAXS analysis.Following SAXS data reduction, Guinier, Kratky, and Pair-distance [P(r)] distribution analyses were performed, analyses that indicated a flexible and dynamic GST-TrbB WT .Consequently, the Ensemble Optimization Method (EOM) was employed to obtain a better understanding of the protein's structural flexibility and dynamics (Da Vela and Svergun, 2020;Tossavainen et al., 2018;Tria et al., 2015).
SAXS provides structural information from signal-averaged X-ray scattering of the protein as it freely diffuses, interacts with solvent molecules, and occupies 3D space in solution (Fig. 4A), meaning the dynamics of the protein in solution can be inferred from the data ( Jacques and Trewhella, 2010;Putnam et al., 2007).The reliability of the collected SAXS data is backed by well-fit Guinier (Fig. 4B, r 2 = 0.99) and P(r) distribution (Fig. 4C, χ 2 = 0.93).Further, the radius of gyration (R g ) values from the Guinier (40.41 ± 0.14 Å) and P(r) from GNOM (43.14 ± 0.27 Å) analyses are within an agreeable range (Table 1).The R g is the weighted root mean square of the intramolecular distances with respect to the centroid of the electron density, effectively quantitating the size of the macromolecule in solution (Da Vela and Svergun, 2020).In other words, R g is a radial averaging of all the conformations sampled by the macromolecule in solution.
The observed molecular weight (MW) from SEC-MALS (86.0 kDa; Supplementary Fig. S3) and SAXS (Vp: 119.9 kDa; Vc: 102.6 kDa; Shape and Size: 106.4 kDa; and Bayes: 109.1 kDa) are over-estimated compared to the theoretical MW of a monomeric GST-TrbB WT (46.4 kDa; Supplementary Fig. S3).The experimental MW values are closer to the theoretical MW value of a homodimer (92.8 kDa).The differential refractive index (dRI) trace (blue) from SEC-MALS does not suggest the presence of large protein aggregates; instead, it indicates the elution of a homogenous fraction (Graewert et al., 2020;Some et al., 2019).The MW estimate (red) across the dRI peak indicates the presence of a dimeric construct (86.0 kDa, Supplementary Fig. S3).
Kratky analysis of the SAXS data also supports that GST-TrbB WT is partially disordered.Deviations from the coordinates (1.732, 1.104) in the Kratky plot (Fig. 4D, marked by red-dashed cross) indicate structural disorder (Da Vela and Svergun, 2020).A partially disordered protein is often indicated by a bell-shaped Gaussian peak that gradually returns to the baseline (Rambo and Tainer, 2013); this is observed in the Kratky plot (Fig. 4D).Accordingly, SAXS bead models are often utilized with high-resolution models, such as that solved by X-ray crystallography or NMR, to determine whether the protein adopts a wider range of conformations that high-resolution methods cannot determine (Kikhney and Svergun, 2015;Powers et al., 2019).TrbB WT has evaded high-resolution structure characterization, but the SAXS (Kratky plot, Fig. 4D), CF-AF2 (Fig. 1B), and 1 H-15 N HSQC spectroscopy (Fig. 3, pronounced clustering of TrbB WT [magenta] 1 H resonances about 8-8.5 ppm; Section 3.1) indicate that TrbB WT is partially disordered (Fig. 1A-C; Supplementary Figs.S1 and S2C), and that the protein is dynamic based on its over-estimated R g values (Fig. 4B and C; Table 1).These insights informed the design of the truncation mutant, TrbB 37-161 .Based on the normalized Pair-distance P(r) distribution function (Fig. 4C), GST-TrbB WT has an elongated shape as opposed to globular (bell-shaped Gaussian), dumbbell (bimodal), or a core-shell (leading asymmetric peak close to the D max ) (Da Vela and Svergun, 2020).The P (r) curve (Fig. 4C) is observed as a tailing asymmetric peak marked by a modest second peak mid r, and gradually approaches 0 at high r, suggesting an elongated shape.
EOM was employed to generate models for GST-TrbB WT with the disordered N-terminal TrbB WT treated as a flexible linker.The EOM χ 2 = 0.88 (Fig. 4E) indicates that the discrepancy between models from the selected ensemble and empirical SAXS data is low (χ 2 → 1 is ideal; Franke et al., 2015;Tossavainen et al., 2018;Tria et al., 2015).The R flex for the selected ensemble (67.42%,Fig. 4F) indicates a significant degree of conformational polydispersity, supporting the inference of a dynamic GST-TrbB WT .The quantitative parameters in Fig. 4F show the lower R flex of the selected ensemble (R flex = 67.42%)compared to that of the pool of conformational possibilities (R flex = 83.63%),indicating a high degree of conformational polydispersity.The ratio of the standard deviation among conformations in the selected ensemble and the standard deviation among conformations in the pool of random possible conformations is described by R σ .Since R σ = 0.98, the standard deviation of the conformations in the selected ensemble (numerator) is modestly lower compared to that of the pool of possible conformations (denominator), meaning there are more conformations possible than in the selected ensemble modelled in Fig. 4G (Da Vela and Svergun, 2020;Tria et al., 2015).Overall, GST-TrbB WT is highly dynamic due to its high degree of conformational polydispersity.Fig. 4G diagrammatically summarizes the SAXS/EOM analyses, illustrating the central role of the N-terminus in the chimeric protein's dynamics (Fig. 4G green surface representation).Changes in D max and R g represented by the structural models illustrate GST-TrbB WT 's conformational dynamics.Due to the disordered N-terminus (residues S1-R36 according to CF-AF2, Fig. 1A-C), GST-TrbB WT is intramolecularly flexible, adopting five representative conformations from a compact and well-folded three-body system (Fig. 4G: Model 1) to a disordered and dispersed system (Fig. 4G: Model 5).The other three structural models (Fig. 4G: Models 2-4) are intermediate conformations that are observed to be less compact compared to Model 1.In considering the fractional contribution of each conformer, GST-TrbB WT appears to adopt conformer 1 at a large probability (Fig. 4G: Model 1, Fraction 5/9), while the other four conformers are each only adopted at a smaller probability (Fig. 4G: Models 1, 3-5, Fraction 1/9).
From a dynamic equilibrium perspective, the greater probability of conformer 1 existing suggests that it is likely the most transiently stable conformer.This model (Fig. 4G: Model 1) indicates that two TrbB 37-161 molecules are well-packed with each other.CORAL indicates that the point-of-contact between two GST-TrbB WT is through TrbB 37-161 (χ 2 = 1.41,Supplementary Fig. S4C), which is supported by the improved EOM fit (χ 2 = 0.97, Supplementary Fig. S5A; 0.9 ≤ χ 2 ≤ 1.1 is ideal; Franke et al., 2015) compared to that which is linked through GST moieties (χ 2 = 0.88, Supplementary Fig. S5B).This result is likely a phenomenon caused by two factors; firstly, a large proportion of GST-TrbB molecules in solution likely have both GST and TrbB 37-161 in a well-packed interface such that CORAL and EOM classify them as both forming a dimer, when the two TrbB 37-161 molecules could simply be within very close proximity at a given moment due to their dynamic folding/unfolding cycles.TrbB's dynamic intramolecular flexibility likely contributes to its function as a DI.The second is that GST may have some structural disorder at its C-terminal when it is part of a chimeric construct with TrbB WT (Kuhnert et al., 2005;Smyth et al., 2003); as both EOM and CORAL are using data and models that fit well with 4 bodies of which two are connected, the result may fit better with a smaller GST model dimerized.Hence, when the TrbB 37-161 dimer (smaller MW vs. that of GST) is specified as the interface for contact in the CORAL and EOM experimental parameters, a better fit of the output model to the scattering curve is observed.The EOM and CORAL results indicate that in the context of a GST-TrbB dimer, TrbB can adopt a well-packed state transiently, providing some evidence for why untagged TrbB is prone to aggregation and thereby difficult to study in vitro using methods that require high protein concentrations, such as X-ray crystallography or SAXS.
When R g values (Fig. 4G) are arranged in an ascending order, the pattern from the D max values surprisingly do not correlate with the R g ascending trend (Supplementary Fig. S6).While R g values have an ascending trend (Model 1 to 5), D max values appear to increase (Model 1 to 3), decrease (Model 3 to 4), then increase (Model 4 to 5); this can be attributed to the difference in dynamics of one locale of the protein A.J. Apostol et al.
(indicated by R g ) compared to the dynamics of the whole protein (indicated by D max ), providing additional evidence of intramolecular flexibility.Notably, model 1 shows the most abundant conformation (Fraction: 5/9, Fig. 4G) and TrbB WT (magenta) appears to be in its most compact state compared to other conformers (Model 2 to 5).The fourbody system is also generally compact.The significant jumps in D max between conformers (Model 1 to 2, ΔD max = 31.10Å, a ~26% increase; Model 2 to 3, ΔD max = 66.70 Å, a ~44% increase; Model 3 to 4, ΔD max = − 48.10 Å, a ~22% decrease; Model 4 to 5, ΔD max = 61.50Å, a ~36% increase) support the flexibility of GST-TrbB WT owing to the N-terminus (Fig. 4G, green) and the fold of TrbB 37-161 as it interacts with GST (observe the three-body system going from compact to dispersed across the five models, Fig. 4G) and with itself (observe magenta changing conformations across the five models, Fig. 4G).In comparing conformer 1 (Model 1, D max = 119.40Å, Fig. 4G) and conformer 5 (Model 5, D max = 230.60Å, Fig. 4G), a 93% increase in D max is observed, explained by the transition from a compact three-body system to a well-dispersed system, and the changes in the observed D max is caused by the changes in the flexibility of the N-terminus (green) and the intramolecular fold of TrbB 37-161 (magenta).

The disordered N-terminus is not required for disulfide isomerase activity in vitro
The effects of N-terminal deletion on the enzymatic activity of TrbB in vitro was investigated.First, we re-established that TrbB WT does function as a disulfide isomerase (DI) in vitro compared to Bovine Serum Albumin (BSA), known not to function as a DI, to a statistically significant difference (p < 0.0001) (Fig. 5A; Supplementary Table S1).Second, the truncation of the N-terminal region of TrbB WT (S1-R36, Fig. 1A-B) was shown to have no effect on the DI activity of the protein in vitro as observed by assaying 50 μM TrbB 37-161 compared to 50 μM BSA (Fig. 5B; Supplementary Table S1); a statistically significant difference was maintained (p < 0.0001).Comparison between TrbB WT and TrbB 37-161 indicated no significant difference (p = 0.76) between the two protein constructs (Fig. 5C; Supplementary Table S1).This data is consistent with the CF-AF2 model and previously reported data suggesting a disordered N-terminus of TrbB WT (Hemmis et al., 2011).The data herein suggests that the thioredoxin domain of TrbB is sufficient for DI activity, and the disordered N-terminal region of the protein does not play a role in TrbB's function, supporting previous findings from studies that determined the effect of mutating the active site residues (Elton et al., 2005;Hemmis et al., 2011).

TrbB does not form a stable complex with TraW in vitro
A previous study by Harris and Silverman (2004) proposed the binding of T4SS F proteins, including TraW and TrbB, using yeast two-hybrid analysis.To test whether TrbB binds TraW as previously reported, TrbB WT and TraW as well as their truncation mutants (TrbB 37-161 and His 6 ΔTraW) were mixed in solution and allowed to equilibrate on ice for at least 30 min and analyzed using SEC (Fig. 6).Elution profiles of individual proteins (TrbB WT , TrbB 37-161 , His 6 TraW, His 6 ΔTraW) were observed as supporting previous sedimentation studies demonstrating that TrbB is monomeric in solution (Hemmis et al., 2011).There is no apparent difference in the elution profiles between the individual proteins and their mixtures (TrbB WT /His 6 TraW, TrbB WT /His 6 ΔTraW), which all elute at ~8 min (Fig. 6A), though there is an increase in the magnitude of the peak absorbances when comparing the protein mixture and individual proteins attributable to the increased protein in solution (Fig. 6A).
TrbB is a disulfide isomerase (this study; Hemmis et al., 2011;Elton et al., 2005;Frost et al., 1994), and is involved in the proper formation of disulfide bonds.TraW, with only one cysteine residue, does not have any potential to form any intramolecular disulfide bonds.However, TraW may be a client protein to a disulfide isomerase such as TrbB if it requires guided redox reactions for forming intermolecular cystines.We investigated whether it was important for TrbB to be reduced to bind TraW as TrbB is reported to be active only in its reduced form (Hemmis et al., 2011).In the cell, DsbD is important for the catalytic activity of TrbB; DsbD's role is to reduce TrbB.In lieu of DsbD, TrbB was reduced using 2 mM DTT as previously done to assay the activity of DsbC (Chen et al., 1999).However, TrbB, even in its reduced form, does not bind TraW (Fig. 6B).
We modelled the predicted TrbB-TraW complex to further understand the putative binding interaction between TrbB and TraW.The computational model shows that TrbB WT and TraW could indeed interact (Fig. 6C top), although with a high predicted alignment error (PAE) (Fig. 6C bottom), and a low predicted template modelling score (pTM, 0.48), interface pTM (ipTM, 0.19) and confidence (a weighted combination of ipTM and pTM) of 0.25 (Evans et al., 2021).The model predicts that the N-terminal residues of TrbB WT are involved in binding N-terminus of TraW, and that TrbB's CXXC motif in its thioredoxin domain is not located at the binding interface (Fig. 6C).Therefore, while TrbB likely functions as a protein chaperone for other T4SS F proteins (Bragagnolo et al., 2020;Elton et al., 2005;Frost et al., 1994), it does not bind TraW in vitro.The difference between our current study (Fig. 6A-B) and that of Harris and Silverman (2004) requires further empirical investigations to ascertain whether TrbB binds TraW.For instance, while yeast two-hybrid analysis is a powerful method to identify protein-protein interactions, high intracellular traffic can lead to non-specific binding and the detection of confounding protein-protein interactions, among other limitations (Huang et al., 2007;Rajagopala et al., 2009;Stellberger et al., 2010).Next, we utilized purified proteins in the current study whereas the proteins may require other conditions in vivo, such as the presence of the lipid membrane or other microenvironment conditions to interact.Our current in vitro binding study employed protein concentrations that may not have been sufficiently high to form an amount of the complex observable in the SEC elution profiles, or even that equilibration times were not sufficient for stable complex formation.Therefore, further studies are on-going to exam the rate at which TrbB binds TraW by an empirical determination of k on (Jarmoskaite et al., 2020) employing orthogonal methods such as Bio-Layer Interferometry to determine the k on , k off , and K D of the TrbB/TraW interaction (Petersen, 2017).It is also important to consider that chaperones are known as weak and transient binders (Arhar et al., 2021), and that some chaperones have dissociation constant (K D ) values in the order of 200 μM (Lee et al., 2015).Understanding this principle is guiding our investigations on TrbB's interactions using methods that can probe transient interactions such as Fluorescence Resonance Energy Transfer (Nguyen et al., 2022).

Conclusions
Disulfide isomerases and other pathways assisting protein folding are critical to ensure proper structural stability and function of proteins.In conjugative T4SSs, like that of the E. coli F plasmid, properly folded proteins and their interactions are critical to effectively affect conjugative DNA transfer from donor to recipient cell.There are several T4SS proteins in F-like plasmids that have high cysteine content, and therefore the plasmid encodes for a disulfide isomerase, TrbB, to aid in maintaining proper protein folding.In the current study, we present a solution characterization of the TrbB protein from the F-T4SS, which contains a disordered N-terminal domain and a C-terminal region containing an active thioredoxin motif.Utilizing CD and 1 H-15 N HSQC-NMR spectroscopies, we show that removal of the N-terminal region of the protein, the TrbB 37-161 construct, results in a more thermally stable and well-ordered protein than TrbB WT (Figs. 1-3).Furthermore, removal of the 36 residue N-terminal region of the protein does not affect the disulfide isomerase (DI) activity of the protein in vitro (Fig. 5).SEC-MALS-SAXS analysis of GST-TrbB WT (Fig. 4) shows a dynamic and dimeric structure, most likely dimerized via the GST tag, that can adopt multiple conformations in solution largely due to the disordered N-terminal region of TrbB; it is this region that provides full-length TrbB with some of its structurally dynamic nature.A previously identified binding partner, TraW, was not observed to interact with TrbB in vitro, although CF-AF2 modelling does suggest that a TrbB-TraW complex is possible, mediated by the N-terminal regions of each protein (Fig. 6).These studies suggest that TrbB employs it's disordered and dynamic N-terminal region as a point of interaction with other T4SS proteins to enable DI activity to ensure proper protein folding, function, and assembly of other F-plasmid proteins to facilitate T4SS F -mediated conjugative DNA transfer.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Structural organization of TrbB WT and TrbB 37-161 .(A) Domain structures based on primary sequence of pre-processed TrbB (signal sequence intact), TrbB WT (following removal of the signal sequence), and the truncation construct TrbB 37-161 .ColabFold-AlphaFold2 predicted 3D models of (B) TrbB WT and (C) TrbB 37-161 .(D) Circular Dichroism spectra expressed in mean residue ellipticity (MRE) of TrbB WT (magenta) and TrbB 37-161 (maroon) were (E) analyzed using BeStSel (Micsonai et al., 2022) to indicate secondary structure composition.CD measurements were collected in triplicate from 5 μM protein samples at 22 • C. For comparison to the empirical data, (F) CF-AF2 secondary structure composition was determined by normalizing number of residues predicted to form α-helices, β-sheets, or loops, by the total number of residues in the protein construct.

Fig. 2 .
Fig. 2. Thermal denaturation profiles of α-helices in (A) TrbB 37-161 and (B) TrbB WT .Circular dichroism at 222 nm expressed in mean residue ellipticity (MRE) of 5 μM protein samples was monitored as a function of temperature.The midpoint of unfolding (T M ) is given by the temperature at which the first derivative of the CD vs T curve is at its highest; T M of TrbB 37-161 is 78 • C (in all replicates), that of TrbB WT cannot be reliably ascertained due to significant data deviations.Data points are shown as mean ± SD, n = 3 internal replicates.

Fig. 3 .
Fig. 3. N-terminal truncation of TrbB leads to a more stable protein.Shown is an overlap of the 1 H-15 N Heteronuclear Single Quantum Correlation (HSQC) spectra of TrbB WT (magenta) and TrbB 37-161 (black).Protein samples, at a concentration of 0.2 mM in 10 mM MES pH 6.0, 50 mM NaCl, 10% (v/v) D 2 O, were analyzed at 600 MHz.The increase in well resolved peaks in the TrbB 37-161 spectrum indicates a less structurally dynamic and more stably folded protein.

Fig. 4 .
Fig. 4. SEC-MALS-SAXS analysis of TrbB WT indicates a structurally dynamic protein.(A) SAXS scattering profile, (B) Guinier fit analysis, (C) Pair-distance [P(r)] distribution analysis from GNOM, and (D) Kratky analysis of 5.5 mg/ml GST-TrbB WT .(E) The Ensemble Optimization Method (EOM) was performed, with the goodness-of-fit metric displayed, to further analyze the structural dynamics of the protein.(F) The EOM-generated distribution of the randomly generated conformations (pool, blue curve) and the selected ensemble that fit the SAXS data (orange curve) are shown, along with metrics of conformational disorder (R flex , R σ ).(G) Models from EOM-SAXS analysis showing GST-TrbB WT 's conformational polydispersity, colour-coded to emphasize the GST moiety (red), the disordered N-terminus of TrbB WT (green), and TrbB 37-161 (magenta).(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)